The broad long-term objective of this proposed project is to uncover the pathophysiological mechanisms of human auditory diseases by analyzing mouse genetic models using genomic and proteomic approaches. Few protein-profiling studies of mouse ear tissues have been attempted because of the difficulty of accessing the small numbers of diverse cells within the hard temporal bone that encases the inner ear. To address this problem, a subtractive strategy was devised in which the digitized abundance of transcripts and proteins in mutant mice with lost inner ear structures is subtracted from those of control mice with normal inner structures. For example, a predominate histological feature of the ears of mice homozygous for the dreidel (ddl) mutation of Pou4f3 is a lack of hair cells in the organ of Corti (OC). Therefore transcripts and proteins of hair cells will be revealed by subtraction of the OC contents of ddl/ddl mutants from those of control mice. The subtractive digitized comparisons of proteome content will be performed using the newly developed two-dimensional difference in gel electrophoresis (2D DICE) method for protein separation and mass spectrometry (MS) for protein identification. Transcript comparisons between mutants and controls will be made by cDNA microarray analysis. Immunohistochemistry and real time RT- PCR approaches will be used to validate protein and mRNA expression patterns and confirm inner ear localization of genes that are identified by 2D DIGE and MS. In this way, a catalog of transcriptome and proteome contents of particular inner ear structures will be compiled for mice at different ages, and an online database will be established to execute a data-sharing plan. This proposal is in response to the NIH Program Announcement PA-03-151 "Proteomics in Auditory Developmental and Disease Processes." The relevance of the proposed research to public health: Techniques developed and data to be collected in this project will be useful for developing strategies for prevention and treatment of human ear diseases. Genes, proteins and their molecular pathways identified in this proposed research are potential targets of drug therapies for hearing loss that affects more than 28 million Americans.